The use of bone markers in clinical studies has provided both important insights into physiological processes, and information on therapy for the treatment of metabolic bone disease. Osteocalcin is one of the most extensively studied biological markers of bone turnover, and it is generally regarded as an index of osteoblastic activity. However, the clinical interpretation of an osteocalcin measurement in individual patients is often ambiguous and does not always correlate with other markers, or with clinical findings. These difficulties arise because of differences in assay specificity, uncertainties regarding the origin of the circulating forms of the protein, and lack of a clear understanding of its function in bone. Studies in the applicant laboratory demonstrate that in addition to the intact protein, fragments of osteocalcin are found in the circulation. They have identified several of these fragments by the use of antibodies with specific epitopes. These individual fragments of osteocalcin may be produced by osteoblasts and/or osteoclasts, and may reflect distinct cellular processes related to the function of osteocalcin in bone. In light of this, the proposed experiments are designed to answer the following questions: (1) what are the forms of osteocalcin in the circulation? Efforts are proposed to further identify the circulating forms of osteocalcin by using specific immunoassays coupled with HPLC and mass spectral analysis. The planned experimentation will identify and quantitate these forms (a) in serum from normal adults and children, with respect to age, race, and sex, and (b) in patients with metabolic bone disease. The resultant values will be compared to standard clinical measures, including calcitropic hormones, minerals, markers of bone resorption, and bone density; (2) what is the source of circulating osteocalcin? The applicants will study (a) osteocalcin biosynthesis, using primary cultures of osteoblasts, (b) the catabolism of osteocalcin, using rabbit-derived osteoclasts in a bone resorbing system, and c) metabolic clearance of the major fragments; and (3) what is the function of osteocalcin in bone? Here, the potential role of osteocalcin in bone turnover and calcium homeostasis will be examined by studying the response of osteocalcin-deplete mice to acute and chronic mineral and hormonal alterations. Responsiveness will be determined by standard measures of calcium homeostasis, including calcitropic hormones, minerals, markers of bone resorption, alternative measures of bone formation, bone density, histomorphometry, and mineral quality and content in bone.